profiles are reported as a percentage of the total radioactivity collected during
the entire HPLC run. The relative distributions of radioactive metabolites in
urine, bile, and feces are calculated from the percent of dose excreted in the
matrix (based on DPM by scintillation counting) multiplied by the percent of
distribution of metabolites in chromatograms of the matrix (based on CPM
counting by Top Count).
18.3.5 Metabolite Identification
The structural identification of metabolites can be carried out at multiple levels.
The goal of initial identification of metabolites is often accomplished through
LC/UV, LC/MS, and LC/MS/MS analyses to determine the biotransformation
pathways involved in the clearance of the compound. Determination of the
nature of the metabolites such as oxygenation (hydroxylation or oxidation of a
heteroatom), dioxygenation, dealkylation, reduction, and conjugation (glucur-
onide, sulfate, glutathione, etc.) is often sufficient. The identification of
metabolites from biological matrices beyond this initial level is often challenging.
Due to low concentrations and interference from endogenous components,
identification of metabolites with unusual structures presents additional
challenges. Neutral loss and product ion scans are used for real-time, data-
dependent acquisition of full MS/MS data of expected metabolites to improve
the selectivity and sensitivity.
Following HPLC separation, samples are analyzed by a mass spectrometer
with an ESI source such as an LCQ, LTQ, (ThermoFinnigan, San Jose, CA),
or Q-TOF Ultima mass spectrometer (Micromass, Beverly, MA). Samples are
analyzed in the positive or negative ion mode. The capillary temperature used
for the LCQ and LTQ, the desolvation temperature used on the Q-TOF, the
nitrogen gas flow rate, spray and cone voltages are adjusted to give maximum
sensitivity for the parent compound.
In the muraglitazar study, we have employed accurate mass spectrometry for
the analysis of the metabolite profiles in biological matrices such as human feces
(Zhang et al., 2007). A Q-TOF Ultima mass spectrometer was used. The Q-TOF
was tuned to 18,000 resolution at half peak height using an insulin tuning
solution, and was calibrated up to 1500 Da using a polyalanine calibration solu-
tion. For accurate mass measurement, them/z556.2771 of an infused 20 ng/mL
leucine enkephalin solution were used as lock mass. The experimentally obtained
masses were within 5 mDa compared to their respective calculated values. The
accurate mass measurements not only provided cleaner selective ion chromato-
grams (ion chromatograms extracted with a mass accuracy to the second decimal
places) of metabolites in feces but also provided spectra for easy identification of
the molecular ions, which greatly enhanced the metabolite identification. The
mass defect filtering (MDF) methodology (Zhang et al., 2003) can be used to aid
in the identification molecular ions of metabolites with unusual structures or
present at low concentrations as well as for metabolites formed from expected
biotransformations.
584 ADME STUDIES IN ANIMALS AND HUMANS